ES2510390T3 - COPD diagnosis - Google Patents

Abstract

Procedure for the diagnosis of chronic obstructive pulmonary disease (COPD) in a human subject comprising the steps of: - determining the amount of heat shock protein 27 (HSP27) in a sample of blood, serum or plasma of a human subject, - diagnose COPD when the amount of HSP27 is increased compared to the amount of HSP27 in healthy human subjects.

Description

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DESCRIPTION

COPD diagnosis

The present invention relates to methods for the diagnosis of COPD in a human subject.

Chronic obstructive pulmonary disease (COPD) is one of the leading causes of death worldwide. In 2020, only ischemic heart disease and cerebrovascular diseases will be responsible for a higher mortality among the world population. Prevalence and hospitalization rates have increased dramatically in recent years. Several studies have shown a strong correlation between smoking and the development of COPD although not all smokers develop the clinical features of COPD (Higenbottam T et al. (1980) Lancet 315: 409-411). The pathogenesis is characterized by obstruction of the respiratory flow due to the remodeling of the airways and an aberrant inflammation. COPD includes chronic bronchitis and emphysema, both conditions characterized by tissue destruction. Restriction of respiratory flow progresses slowly, leading to dyspnea and limitations on the ability to exercise. However, the deficiency is not limited to the lungs since COPD patients also have a higher risk of systemic insufficiencies that include cardiovascular disease. The diagnosis of airway obstruction according to the guidelines of the Global Initiative for Chronic Obstructive Pulmonary Diseases (GOLD) requires the use of spirometry. A FEV1 / FVC ratio (forced expiratory volume in a safe / forced vital capacity) postbronchodilator less than 70% indicates an irreversible airway obstruction and, therefore, is considered to be the main parameter for the diagnosis of COPD (Global Strategy for Diagnosis, Management, and Prevention of COPD. Global Initiative FOR Chronic Obstructive Lung Disease, 2007, www.goldcopd.com). Currently, patients are classified within the GOLD stages according to spirometry data and clinical presentation. Detection of serum markers that indicate the activity of the

25 disease is of special interest in the diagnostic and therapeutic process.

Although it is widely accepted that smoking is the main risk factor for the development of COPD, descriptions of the specific pathogenic pathway and the mechanisms involved remain vague. Oxidative stress in the lung parenchyma seems to be involved in the onset of the inflammatory response to exposure to tobacco smoke. Neutrophils and macrophages, as part of innate immunity, were considered fundamental in the airway remodeling process in COPD. This remodeling process is a consequence of the induction of chronic apoptosis in the lungs of patients with COPD or in smokers. The term apoptosis refers to the morphological alteration shown by cells that die "actively" which includes decreased cell volume, membrane blistering, condensation

35 of chromatin and DNA fragmentation. Apoptosis affects the alveolar and bronchial epithelial cells as well as the parenchyma endothelial cells. Controlled apoptosis is a desired and critical process for cell hemostasis. However, excessive triggering of apoptosis and an increase in alveolar cell turnover can lead to tissue destruction.

Immunohistochemical observations in derive lung tissue of patients with COPD showed inflammatory infiltrates composed of T lymphocytes and macrophages, with varying amounts of mast cells, neutrophils and eosinophils. Advances in immune reactions have shown that an imbalance between type 1 helper T lymphocytes (TH1) and type 2 helper T lymphocytes (TH2) has a role in the inflammatory response of various diseases. Studies have shown a predominant pattern of TH1 cytokines in the

45 infiltrating interstitial inflammation in COPD. The airway epithelium is a critical component of the immune reaction, producing proinflammatory cytokines such as tumor necrosis factor alpha (TNFα), interleukin-1-beta (IL-1β) and chemokines. Epithelial lung cells also modulate acute exacerbations and reactions to pathogens that are common in patients with COPD.

The only procedure used in clinical practice for the diagnosis of COPD is spirometry, which is a test of lung function in which said lung function is measured, specifically, the measurement of quantity (volume) and / or velocity ( flow) of air that can be inhaled and exhaled. However, these tests are also used to diagnose other lung diseases such as asthma and pulmonary fibrosis. Therefore, this procedure cannot function as the only test for a reliable diagnosis of COPD. In

Consequently, doctors also consider symptoms such as dyspnea, chronic cough and sputum production and / or a history of exposure to risk factors in the diagnosis of COPD. It is evident that the use of these procedures in the diagnosis of COPD or in the discrimination between various forms of COPD can lead to a false diagnosis, so that the patient cannot use the best form of treatment right from the beginning of the illness.

Therefore, it is an object of the present invention to provide methods and measures that allow an unequivocal diagnosis of COPD in a human subject from the onset of the disease or even determine the risk that a human subject develops COPD.

The present invention relates to a method for diagnosing chronic obstructive pulmonary disease (COPD) in a human subject comprising the steps of:

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-determine the amount of heat shock protein 27 (HSP27) in a sample of blood, serum or plasma from a human subject,

5 -diagnosing COPD when the amount of HSP27 is increased compared to the amount of HSP27 in healthy human subjects.

According to a preferred embodiment of the present invention, in addition, the concentration of interleukin-1 receptor 4, histone-associated DNA fragment (histones), HSP70 and / or HSP90 alpha is increased by

Comparison with the number of 4 interleukin-1 receptors, DNA fragment associated with histones, HSP70 and / or HSP90 alpha in healthy humans.

It was found that the concentration of HSP27, optionally in combination with ccCK-18, histones, HSP70 and / or HSP90 alpha in a sample of a human can function as a marker to diagnose COPD, in particular,

15 stages I / II and III / IV of COPD in said human subject. Markers even determine the risk of a human subject developing COPD in the future. Not all humans considered to be at risk of developing COPD because of their lifestyle (for example, humans exposed to smoke, smokers, etc.) will get COPD. Therefore, the diagnosis that a human subject is at risk of developing COPD is very useful for the prevention of COPD.

The markers used in the process of the present description can be used separately or in combination, whereby additionally the concentration of receptor 4 of interleukin-1 can be determined, in which COPD is diagnosed when the concentration of receptor 4 of interleukin-1 is increased compared to the amount of interleukin-1 receptor 4 in healthy human subjects or in which the risk of

Developing COPD is diagnosed when the amount of interleukin-1 receptor 4 is reduced compared to the amount of interleukin 1 receptor 4 in healthy humans. Preferred combinations include interleukin-1 receptor and ccCK-18, interleukin-1 receptor and histones 4, ccCK-18 and histones, interleukin-1 receptor 4, HSP27, HSP70 and / or HSP90 alpha.

30 To classify the severity of COPD, spirometric parameters are used. These parameters allow the severity of COPD to be classified into four stages (see table A). Spirometry is essential for diagnosis and provides a useful description of the severity of pathological changes in COPD. Specific spirometric cut-off points (for example, post-bronchodilator VEF1 / CVF ratio <0.70 or VEF1 <80, 50 or 30% of the expected) are used to determine stages I to IV of COPD.

35 Table A: COPD spirometric classification (according to www.goldcopd.com) Gravity based on the VEF1 post-bronchodilator.

Stage I: mild

FEV1 / FVC <0.70 FEV1 80% of expected

Stage II: Moderate

FEV1 / FVC <0.70 50% FEV1 <80% of expected

Stage III: serious

FEV1 / FVC <0.70 30% FEV1 <50% of expected

Stage IV: very serious

FEV1 / FVC <0.70 FEV1 <30% of expected or FEV1 <50% of expected more chronic respiratory failure

FEV1: forced expiratory volume in one second; FVC: forced vital capacity; respiratory failure: partial arterial oxygen pressure (PaO2) less than 8.0 kPa (60 mmHg) with or without partial arterial CO2 pressure (PaCO2) greater than 6.7 kPa (50 mmHg) breathing air at sea level.

The procedures for the determination of FEV1 and FVC, which can be used to systematize COPD (see table A) are well known in the art (see for example, Eaton T et al., Chest (1999) 116: 416- 23; Schermer TR, et al., Thorax (2003) 58: 861-6; Bolton CE, et al., Respir Med (2005) 99: 493-500).

45 The effect of COPD on a particular patient depends not only on the degree of limitation of respiratory flow but also on the severity of the symptoms (especially respiratory failure and decreased ability to exercise). There is only an imperfect relationship between the degree of limitation of respiratory flow and the presence of symptoms. The characteristic symptoms of COPD are dyspnea, cough and chronic and progressive sputum production. Cough and chronic sputum production may precede the development of respiratory flow limitation

50 in many years. This pattern offers a unique opportunity to identify smokers and other people at risk

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COPD, and intervene when the disease is not yet a health problem.

On the contrary, it can develop a significant limitation of respiratory flow without cough and chronic sputum production. Although COPD is defined based on the limitation of respiratory flow, in practice the decision to go to the doctor (and thus allow the diagnosis to be made) is usually determined by the effect of a particular symptom on the style of the patient's life.

Stage I: Mild COPD - characterized by a slight limitation of respiratory flow (FEV1 / FVC <0.70; FEV1 80% of expected). Symptoms of chronic cough and sputum production may occur, but not always. In this stadium, the

10 individual normally does not know that his lung function is abnormal.

Stage II: Moderate COPD - characterized by a worsening of the respiratory flow limitation (FEV1 / FVC <0.70; 50% FEV1 <80% than expected), with difficulty breathing typically develops during exercise and occasionally appears cough and sputum production. This is the stage where patients typically go to

15 doctor due to chronic respiratory symptoms and an exacerbation of his illness.

Stage III: Severe COPD - characterized by further worsening of respiratory flow limitation (FEV1 / FVC <0.70; 30% FEV1 <50% of expected), major respiratory distress, reduced ability to exercise, fatigue and repeated exacerbations that almost always affect the quality of life of patients.

20 Stage IV: Very severe COPD - characterized by a severe respiratory flow limitation (FEV1 / FVC <0.70; FEV1 <30% of expected or FEV1 <50% of expected more presence of chronic respiratory failure). Respiratory insufficiency is defined as a partial pressure of O2 in arterial blood (PaO2) less than 8.0 kPa (60 mmHg) with or without partial pressure of CO2 in arterial blood (PaCO2) greater than 6.7 kPa (50 mmHg) breathing air at sea level.

25 Respiratory failure can also cause effects on the heart such as pulmonary heart disease (right heart failure). Clinical signs of pulmonary heart disease include elevation of jugular venous pressure and ankle edema with fovea. Patients may present with stage IV: very severe COPD even if FEV1 is> 30% of that expected, provided these complications appear. At this stage, the quality of life is very appreciably altered and exacerbations can pose a threat to life.

30 The term "at risk of developing COPD," as used herein, refers to a group of human subjects who are subject to environmental threats or who may present a genetic predisposition to develop COPD. Factors that promote the onset of COPD include genetic predisposition, exposure to particles such as tobacco smoke, workplace dusts (organic and inorganic),

35 indoor air pollution due to heating and cooking with biomass in poorly ventilated homes and outdoor air pollution, growth and lung development, oxidative stress, sex, age, respiratory infections, socioeconomic status, nutrition and comorbidities. Humans who are at risk of developing COPD may suffer from chronic cough, chronic sputum production and normal spirometry. However, human subjects suffering from these symptoms do not necessarily develop stage I of COPD.

40 As used herein, the term "healthy human subject" refers to humans who do not have COPD or any other lung disease. Additionally, these humans have not suffered any serious lung disease in their life. "Healthy humans" also does not include humans who are regularly exposed to risk factors, such as fumes or harmful substances.

45 According to the present disclosure, the number of markers in "healthy human subjects" is determined by quantifying these markers in at least 5, 10, 15 or 20 "healthy human subjects."

Since the COPD markers determined with the procedure according to the present description are

50 found in the blood, the sample to be used according to the present description is blood, serum or plasma. These types of samples can be obtained from a human with procedures known in the art.

To specifically determine the amount of interleukin-1 receptor 4, cytokeratin-18 cleaved by caspases (ccCK-18), thermal shock protein 27 (HSP27), thermal shock protein 70 (HSP70) and / or protein

55 heat shock 90 alpha (HSP90 alpha) in a sample it is preferred to use methods that use antibodies directed against said COPD markers. Suitable procedures using antibodies are immunoassays that are preferably selected from the group consisting of enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and immunoblot.

The term "antibody", as used herein, refers to monoclonal and polyclonal antibodies or fragments thereof capable of binding to an antigen. Other antibodies and antibody fragments, such as recombinant antibodies, chimeric antibodies, humanized antibodies, antibody fragments such as Fab or Fv fragments, as well as the fragments selected by selection of phage display libraries and similar molecules, are also useful in the described procedures. in this

65 document.

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The procedures for the preparation of monoclonal as well as polyclonal antibodies are well established (Harlow E. et al., 1988. Antibodies. New York: Cold Spring Harbor Laboratory). Polyclonal antibodies are obtained in various species that include, among others, mice, rats, rabbits, goats, sheep, donkeys, camels and horses, using conventional immunization and bleeding procedures. Bleeds of animals with values

High levels are fractionated by routine selective salt addition procedures, such as precipitation with ammonium sulfate, and by separating specific immunoglobulin fractions by successive affinity chromatography on Sepharose-protein A and Sepharose-leptin columns, according to conventional procedures. Next, the specificity of both polyclonal and purified monoclonal antibodies is characterized. This characterization is performed by conventional procedures using proteins labeled with a tracer such as a radioisotope or biotin in competition with increasing levels of possible cross-reactive reagents not labeled for antibody binding. Binding studies are further evaluated by other conventional procedures such as the well-established polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (PAGE-SDS) and immunoblotting procedures under reducing or non-reducing conditions.

15 Monoclonal antibodies are prepared according to well established conventional laboratory procedures ("Practice and Theory of Enzyme Immunoassays" by P. Tijssen [in Laboratory Techniques in Biochemistry and Molecular Biology, Eds: RH Burdon and PH van Kinppenberg; Elsevier Publishers Biomedical Division, 1985]), which are based on the original technique of Kohler and Milstein (Kohler G., Milstein C. Nature 256: 495, 1975). This technique is performed by extracting the thymus cells from immunized animals and immortalizing the cells that produce antibodies by fusion with myeloma cells or by transformation with the Epstein-Barr virus and then selected clones expressing the antibody. desired, although other techniques known in the art are also used. Antibodies are also obtained by other techniques known to those skilled in the art, including but not limited to, immunization with specific DNA.

The immunoassays preferably used are based on techniques in which capture antibodies are used, which are capable of specifically binding to an antigen of interest, or to antigens bound to a solid support. The capture antibody is conjugated or bound to various solid phase supports using conventional covalent or non-covalent binding procedures, depending on the necessary analytical and / or solid phase separation requirements. The solid support is in the form of test tubes, beads, microparticles, filter paper, membranes, glass filters, magnetic particles, glass or silicone chips or other materials and techniques known to those skilled in the art. The use of microparticles, especially magnetizable particles, that have been directly coated with the antibody (magnetic particle capture antibody) or particles that have been labeled with a universal adhesive (eg, avidin or antispecies antibody) is useful due to a reduction

35 significant incubation time of the assay. These together with other alternative techniques known in the art allow the test to be carried out in minutes without limiting the necessary sensitivity.

The detection antibody, which is capable of specifically binding to the antigen of interest, used for the detection of the antigen is directly linked to an indicator molecule, or is detected indirectly by a secondary detection system. The latter is based on several different principles known in the art, which include the recognition of antibodies by means of a labeled anti-species antibody and other forms of immunological or non-immunological linkages and signal amplification detection systems (eg, biotin-streptavidin technology) . The signal amplification technique is used to significantly increase the sensitivity of the assay and the low level of reproducibility and performance. The marker used for direct conjugation or

Indirect to the antibody is any detectable indicator molecule. Examples of suitable markers include those widely used in the field of immunological and non-immunological detection systems, such as fluorophores, luminescent labels, metal complexes and radioactive labels, as well as moieties that could be detected by other suitable reagents such as enzymes, or various combinations of direct or indirect markers such as enzymes with luminogenic substrates.

To diagnose COPD or the risk of developing COPD, it is an advantage to define cut-off levels above or below which the disease can be diagnosed. The concentration of interleukin 1 receptor 4 in blood samples of a healthy human subject preferably ranges from 50 to 150 pg / ml, preferably between 60 and 140 pg / ml, more preferably between 70 and 130 pg / ml.

According to another embodiment of the present description, the concentration of cytokeratin-18 cleaved by caspases (ccCK-18) in blood samples of a healthy human subject ranges between 200 and 350 U / l, preferably between 200 and 330 U / l, more preferably between 250 and 300 U / l, measured as a new epitope M30 of cytokeratin-18.

According to another embodiment of the present description, the concentration of heat shock protein 27 (HSP27), in blood samples of a healthy human subject ranges between 1500 and 2500 pg / ml, preferably between 1600 and 2400 pg / ml, more preferably between 1700 and 2300 pg / ml.

According to another embodiment of the present description, the concentration of thermal shock protein 70 (HSP70), in

65 blood samples from a healthy human subject range from 50 to 200 pg / ml, preferably between 70 and 190 pg / ml, more preferably between 80 and 180 pg / ml.

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According to another embodiment of the present description, the concentration of high heat shock protein 90 (HSP90 alpha), in blood samples of a healthy human subject ranges between 10,000 and 15,000 pg / ml, preferably between 11,000 and 14,500 pg / ml, more preferably between 12,000 and 14,000 pg / ml.

5 According to a further embodiment of the present description the concentration of histones in blood samples of a healthy human subject is 20 to 50% lower, preferably 25 to 40% lower, than in blood samples of a human subject suffering from the COPD

According to another preferred embodiment of the present description, COPD is diagnosed when the concentration of interleukin-1 receptor 4, cytokeratin-18 cleaved by caspases (ccCK-18), histones, HSP27, HSP70 and / or HSP90 alpha in the sample it is increased by at least 10%, preferably at least 20%, compared to the amount of ST2, ccCK-18, histones, HSP27, HSP70 and / or HSP90 alpha in healthy human subjects. Of course the amount of ST2, ccCK-18, histones, HSP27, HSP70 and / or HSP90 alpha can also be increased by at least the

15 15%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.

The risk of developing COPD is preferably diagnosed when the concentration of receptor 4 of interleukin-1, cytokeratin-18 cleaved by caspase (ccCK-18) and / or histones in the sample is reduced by at least 10%, preferably at least 20%, compared to the amount of interleukin receptor 4, ccCK-18 and / or histones in healthy human subjects. Of course, the amount of interleukin-1 receptor 4, ccCK-18 and / or histones can also be reduced by at least 15%, 25%, 30%, 40%, 50%, 60%, 70%, 80 %, 90% or 100%.

The process according to the present description also applies to other types of mammals where the mammal is preferably selected from the group consisting of horse, dog, cat and cattle.

Another aspect of the present invention relates to a method for discriminating between COPD in stages I / II and COPD in stages III / IV in a human subject comprising the steps of:

- determine the concentration of heat shock protein 27 (HSP27) in a blood, serum or plasma sample of a human suffering from COPD,

-diagnosing COPD in stages I / II when the amount of HSP27 is reduced compared to the amount of HPS27 determined in a sample of a human subject suffering from stage III / IV COPD, or

35 -diagnosing COPD in stages III / IV when the amount of HSP27 is increased compared to the amount of HPS27 determined in a sample of a human subject suffering from stage I / II COPD.

According to a particularly preferred embodiment of the present invention, additionally, the concentration of interleukin-1 or HSP70 receptor 4 determined in a sample of a human subject suffering from COPD in stages III / IV and / or when the concentration of ccCK- 18 or histone associated DNA fragment is reduced compared to the concentration of ccCK-18 or histone associated DNA fragment determined in a sample of a human subject suffering from stage III / IV COPD, or where COPD is diagnosed in stages III / IV when the amount of interleukin-1 or HSP70 receptor 4 is reduced compared to the

45 concentration of interleukin-1 receptor 4 determined in a sample of a human subject suffering from COPD in stages I / II and / or when the concentration of ccCK-18 or histones is increased compared to the concentration of ccCK-18 or histone-associated DNA fragment determined in a sample of a human subject suffering from COPD in stages I / II.

The COPD markers described in this document are also suitable to discriminate between humans suffering from stage I / II COPD or stage III / IV COPD as defined above. Discrimination between these stages of COPD is important in determining treatment. In patients with few symptoms or intermittent symptoms (stages I and II), for example, the use of an inhaled bronchodilator of short action is sufficient if necessary to control dyspnea. If inhaled bronchodilators are not available, you should

55 be considered the usual treatment with slow-release theophylline. In humans whose dyspnea during their daily activity is not relieved despite treatment with short-acting bronchodilators as needed, it is recommended to add regular treatment with a long-acting inhaled bronchodilator. In humans with stage III / IV COPD, regular treatment with inhaled glucocorticoids reduces the frequency of exacerbations and improves health status. In these humans, the usual treatment with an inhaled glucocorticoid should be added to long-acting inhaled bronchodilators. In humans with stage III / IV COPD, surgical and / or long-term oxygen treatments should be considered if chronic respiratory failure occurs.

The reference values that allow discriminating between COPD in stages I / II and III / IV can be assessed by

65 the determination of the respective concentrations of interleukin-1 receptor 4, ccCK-18, histones, HSP27 and / or HSP70 in a set of samples obtained from humans suffering from COPD in stages I / II and III / IV .

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This set may comprise samples obtained from at least 5, preferably at least 10, more preferably at least 20, humans suffering from COPD and in which the various stages of COPD have been diagnosed by alternative procedures (eg spirometry ).

5 COPD can be diagnosed in stages I / II in a human subject when the concentration of HSP27 in a sample of said subject ranges between 2600 and 3300 pg / ml, preferably between 2700 and 3200 pg / ml. If the amount of HSP27 in a sample of a human subject ranges between 3400 and 5500 pg / ml, preferably between 3500 and 5000 pg / ml, more preferably between 3600 and 4500 pg / ml, stage III / IV COPD is diagnosed.

COPD can be diagnosed in stages I / II in a human subject when the concentration of interleukin-1 receptor 4 in a sample of said subject ranges between 160 and 400 pg / ml, preferably between 170 and 380 pg / ml, more preferably between 180 and 350 pg / ml. COPD can also be diagnosed in stages I / II when the concentration of receptor 4 of interleukin-1 is more than 160, preferably more than 170, more preferably more than 180 pg / ml in said sample.

15 COPD in stages I / II and COPD in stages III / IV can be diagnosed when the concentration of HPS70 in a sample of a human subject is at least 250 pg / ml, preferably at least 300 pg / ml.

Stage I / II COPD and stage III / IV COPD can be diagnosed when the concentration of HPS90 alpha in a sample of a human subject is at least 15,000 pg / ml, preferably at least 16,000 pg / ml.

The markers shown in this document can also be used to monitor the progress of COPD and the progress of a treatment for COPD. These procedures involve the comparison of the concentration of ccCK-18, histones, HSP27 and / or HSP70 in samples obtained from a human at different time intervals. The

25 results obtained from said procedure allow the doctor to establish an appropriate therapy.

The present description is further illustrated by the following figures, examples and embodiments without this, however, being a restriction.

Figure 1 shows serum concentrations of cytokeratin-18 cleaved by soluble apoptosis specific caspase detected using an M30 antibody (+/- ETM).

The measured optical density (D.O.) values of DNA fragments associated with circulating histones in serum samples (+/- ETM) are shown in Figure 2.

35 Figure 3 shows caspase-1 / ICE levels in the peripheral serum samples of the study patients (+/- ETM).

Figure 4 shows the serum concentrations of maximum soluble ST2 in patients with COPD in stages I and II (+/- ETM).

Figure 5 indicates that IL-10 in serum samples showed no differences between the study groups (+/- ETM).

Figure 6 shows the correlations between serum concentrations of ICE and the number of packages-years

45 of the patients (A), DNA fragments associated with circulating histones and serum levels of ICE (B), FEV1 / CVF and cytokeratin-18 cleaved by serum caspases (C) and anti-inflammatory markers ST2 and IL-10 (D) respectively. R: Spearman rank correlation coefficient.

Figure 7 (a, b, c, d, e, f) shows the serum levels of thermal shock proteins and 20S proteasomes determined in the systemic bloodstream of patients and controls. The results are expressed as the mean ± SEM.

Figure 8a through 8d shows non-parametric correlations between HSP27, HSP70 and inflammatory cytokine IL-6 and the pulmonary function parameter VEF1% CV. R: Correlation coefficient.

55 Figure 9 shows a receiver operating characteristic curve (ROC) indicating sensitivity and specificity of HSP27 and HSP70 to diagnose COPD in the smoking study population.

EXAMPLES:

Example 1:

Materials and methods.

65

Patients

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A total of 64 patients will be included in this case-control study. Healthy volunteers (n = 15), smokers without COPD (n = 14), patients with mild to moderate COPD (n = 19) and patients with severe or very severe COPD (n = 16) were evaluated in four groups of the study. The characteristics of the patients are shown in table 1.

Table 1

Clinical features (the severity of airflow obstruction was determined using pulmonary function tests [PFP] in all subjects; COPD patients who meet the GOLD diagnostic criteria for COPD). The data is provided as the mean (+/- standard deviation) if nothing else is indicated.

Subject Category

Healthy Healthy smoker COPD GOLD I-IV COPD GOLD I and II COPD GOLD III and IV

N

fifteen 14 35 19 16

Man / woman

10/5 7/7 20/15 10/9 10/6

Age

57.20 56.64 59.60 60.68 58.31

Pulmonary function

-

CVF (L)

4.55 3.84 2.80 3.33 2.14

FEV1 (%)

105.37 94.40 52.76 70.21 30.67

FEV1 / CV (%)

76.80 75.95 51.18 61.74 37.80

EMF 50 (%)

100.67 87.64 27.29 39.42 11.93

EMF 25 (%)

103.53 75.71 29.71 37.37 20.00

Smoking history

Never smoked

fifteen 0 0 0 0

Former smoker

0 3 7 4 3

Current smoker

0 eleven 28 fifteen 13

Packagesyears

0 34 (25.2) 45.8 (30.6) 47.3 (29.7) 44.0 (32.6)

Body weight (kg)

71.6 (13.9) 76.4 (8.6) 80.4 (21.6) 79.7 (16.7) 81.1 (27.2)

Height (cm)

172.7 (10.9) 168.7 (8.1) 169.2 (10.5) 167.7 (12.1) 171.2 (7.9)

Abbreviations used: COPD: chronic obstructive pulmonary disease; FEV1: forced expiratory volume in 1

10 second; FVC: Forced vital capacity, GOLD: Global initiative for chronic obstructive pulmonary disease, FEM: Maximum expiratory flow.

Exclusion criteria were acute exacerbation as defined in the WHO guidelines and the Global Initiative for Chronic Obstructive Pulmonary Disease (GOLD, www.goldcopd.com) or drug use 15 immunomodulators in the last 14 days, history of asthma , autoimmune diseases, other relevant lung diseases (for example, lung cancer, known antitrypsin deficiency) or any known cardiopulmonary comorbidity. Height and weight (Seca; Vogel and Halke, Germany) were measured and body mass index (BMI) was determined. The lung function parameters (FEV1, FVC, FEV1: FVC ratio) were measured using the same spirometer model (AutoboxV6200, SensorMedics, Austria). The 20 measurements were made before and, if the requirements for airflow obstruction were met, 15 to 30 minutes after inhaling 200 µg of salbutamol. Arterial gasometry (PaO2, PaCO2) was obtained at rest while breathing the room air in a sitting position. The gasometry measurement was performed with an ABL 510 gas analyzer (Radiometer, Denmark). The results are expressed as absolute values and percentages of expected values for age, sex and height, according to the prediction equations of the European Community for

25 Steel and Coal (European Coal and Steel Community; Quanje PH et al. Eur Respir J Suppl. 16 (1993): 5-40). The normal expected values were derived from the reference values of the Austrian Society of Pulmonary Medicine (Harnoncourt K et al., Österreich Ärztetg. 37 (1982): 1640-1642). Blood samples were taken at the time of lung evaluation. The serum was obtained after centrifugation and the aliquots were kept frozen at -20 ° C until further testing.

30

Quantification of cytokeratin-18 cleaved by caspases (ccCK-18)

Cytokeratin-18 M30 neoepitope levels were measured in samples using the M30-Apoptosense ELISA (Peviva, Sweden). In summary, this ELISA uses an antibody that recognizes a neoepitope exposed after excision induced by cytokeratin apoptosis18. M30 antigen levels can be measured in units per liter (U / l) (1 U / l is equivalent to 1.24 pmoles of a peptide synthesized from the M30 recognition motif) according to the

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maker. It was established that the sensitivity of ELISA was 25 U / l. Intra and inter-assay reproducibility was <10%. Serum concentrations were calculated by comparing the values of D.O. of the samples with the values of D.O. of conventional dilutions.

5 Evaluation of DNA fragments associated with serum histones

A commercial ELISA kit (Roche Applied Science, Germany) was used to quantify the serum content of histone-DNA complexes. Samples were incubated together in 96-well microtiter plates with biotin-conjugated anti-histone mouse monoclonal antibody (clone H11-4) and peroxidated-conjugated anti-DNA mouse monoclonal antibody (clone MCA-33) for two hours at room temperature. After washing the wells, an ABTS solution was added to each well. The enzymatic reaction was monitored until sufficient color developed and the plates were read at 405 nm. The values of D.O. were calculated by subtracting the values of D.O. of the white wells of the average of the sample wells.

15 Quantification of caspase-1 / ICE in serum

An enzyme-linked immunosorbent assay (ELISA) (BenderMedSystems, Austria) was used to determine the soluble contents of soluble caspase-1 / ICE in serum samples. Microtiter plates precoated with monoclonal antibody against human ICE were incubated with the sample material or diluted conventional concentrations (400 pg / ml -6.25 pg / ml, seven dilution steps) at room temperature for 1.5 hours. The plates were washed three times with wash buffer and a rabbit polyclonal anti-ICE antiserum was added for 30 minutes. After another washing step, horseradish peroxidase conjugate was applied for 30 minutes. The wells were washed and a TMB substrate solution was used for the detection of enzymatic activity. The reaction was stopped using sulfuric acid (2N). The plates were read at 450 nm in a

25 Wallac Multilabel 1420 counter (PerkinElmer, USA). The concentrations were calculated by comparing the optical density (D.O.) values of the sample with the D.O. of known concentrations of the standards.

Quantification of interleukin-1 receptor 4

A commercial enzyme-linked immunosorbent assay (ELISA) (R&D Systems, USA) was used to determine the serum contents of interleukin-1 receptor 4 in the samples. The microtiter plates were previously coated with a capture antibody against human ST2. Then the plates were washed and blocked. Samples and standard dilutions (from 2000 pg / ml to 31.25 pg / ml) were incubated at room temperature. A biotinylated human IL-4 goat anti-R4 antibody was used for detection. Be

35 used a TMB substrate solution for the detection of enzyme activity after the addition of streptavidin conjugated with horseradish peroxidase. The reaction was stopped using sulfuric acid (1N). The plates were read at 450 nm in a Wallac Multilabel 1420 counter (PerkinElmer, USA). The concentrations were calculated by comparing the optical density (D.O.) values of the sample with the D.O. of known concentrations of the standards.

Quantification of interleukin-10

An ELISA technique (BenderMesSystems, Austria) was used to quantify the levels of interleukin-10 (IL-10) in serum samples obtained after centrifugation of whole blood. 96-well plates were coated

45 with a monoclonal antibody directed against the specific antigen and incubated overnight at 4 ° C. After a washing step, the plates were blocked with assay buffer for two hours. After another washing step, samples and standards with defined concentrations of the antigen were incubated as described by the manufacturer. Next, the plates were washed and incubated with polyclonal antibodies bound to enzymes. The TMB substrate solution was applied after the appropriate incubation time and another washing step. The color development was then controlled using a Wallac Multilabel 1420 counter (Perkin Elmer, USA). The values of D.O. obtained were compared with the standard curve calculated from the values of D.O. of controls with known concentrations of the antigen.

Statistical methods

55 SPSS software (SPSS Inc., USA) was used to calculate all results. A p value <0.05 was considered statistically significant. Peer comparisons were made between groups using the Mann-Whitney U test. Correlations were calculated using Spearman's correlation coefficient. The results were not corrected for multiple analyzes.

Results

Cytokeratin-18 cleaved by caspases induced by apoptosis is increased in patients with COPD

65 Elevated serum levels of M30 neoepitope were shown in patients with stage I and II COPD (294.96 U / l [87.81-502.11], p = 0.008) and with stage III and IV COPD ( 464.86 U / l [79.69-850.03], p = 0.001) in

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comparison with the serum contents of healthy smokers (119.67 U / l [93.85-145.49]). Healthy controls showed slightly elevated levels of M30 (284.08 U / l [64.26-503.90]), although this difference was not statistically significant (fig. 1).

5 The content of histone-associated DNA fragments was increased in patients with severe or very severe COPD

It was determined that the average value of D.O. in patients with stage III and IV COPD it was 0.43 [0.26-0.60]. This level showed a statistically significant difference with respect to histone levels in sera from healthy controls (0.27 [0.12-0.42], p = 0.037) and smokers without COPD (0.26 [0.08 - 0.45], p = 0.022). Patients with stage I and II COPD had elevated levels of histone-associated DNA fragments (0.40 [0.22-0.59]), although no statistical effect was observed compared to other groups (Fig. 2).

Serum caspase-1 / ICE levels showed no statistically significant differences.

15 ICE concentrations in serum samples were 84.86 pg / ml ([64.85-104.87], with a 95% confidence interval [CI]) for healthy controls and 102.76 pg / ml [ 56.59 -148.93] for smokers without COPD. Patients with COPD in stages GOLD I and II showed a moderately higher average content of 114.56 pg / ml [78.98-150.14] and patients with COPD in stages III and IV had a medium level in 111.87 pg / ml serum [82,21-141,53] comparable to COPD in stages I and II. No significant differences were observed between any of the groups (fig. 3).

Serum levels of interleukin 1 receptor 4 are elevated in patients with COPD

25 High serum levels of interleukin-1 receptor 4 could be demonstrated in patients with stage I and II COPD (251.80 pg / ml [45.74-457.85], p = 0.031) compared to the contents serum of healthy smokers (94.01 pg / ml [11.57-176.46]). In all other groups the comparisons showed no significant differences; healthy controls (99.86 pg / ml [41.86-157.86]), COPD stages III and IV (127.24 pg / ml [51.72 202.75]) (fig. 4).

IL-10 levels showed no statistically significant differences.

The concentrations of IL-10 in serum samples were 8.84 pg / ml ([-6.14 -23.81]) for healthy controls and 10.25 pg / ml [-3.84 -24.34] for smokers without COPD. Patients with COPD in stages GOLD I and II

35 showed a content of 11.57 pg / ml [-8.49 -31.62] and patients with stage III and IV COPD had a lower mean serum level of 0.63 pg / ml [-0, 71 -1.97]. No significant differences were observed between any of the groups (fig. 5).

conclusion

Chronic obstructive disease is characterized by chronic inflammation of the lung tissue and concomitant involvement of other organ systems. This process is considered to be triggered primarily by many years of smoking. A strong correlation between the number of pack-years and the serum concentration of caspase-1 ICE (ICE) proinflammatory could be demonstrated indicating a direct effect of habits

45 smoking on the systemic inflammatory response (fig. 6A). Elevated levels of soluble soluble ccCK-18 apoptosis and histone-associated DNA fragments (histones) could be found in patients with COPD in stages GOLD I and II and GOLD III and IV (Figures 1, 2). Serum contents of biologically active ICE protein correlate significantly with histone levels (Fig. 6B). In apoptotic cells, activation of the endonucleosome causes DNA cleavage and generates long chromatin fragments, short oligonucleosomes rich in H1 and mononucleosomes (called histones) that do not bind to the nucleus. The release of histone-specific DNA apoptosis complexes seems to result in constant remodeling due to immune activation.

The pathogenesis of COPD is associated with degeneration of lung tissue due to increased activation.

55 and proapoptotic signaling of innate and acquired immune systems in patients susceptible to the disease. Chronic inflammation induces an increase in ICE levels, a protease with Cys285 that serves as a catalytic residue, which cleaves the biologically inactive IL-1β precursor of 21 kDa in Asp116-Ala117 to generate the mature 17.5 kDa form of IL-1ß. The active enzyme consists of two non-identical subunits (p10 and p20) both essential for enzymatic activity and, therefore, has a fundamental function in the apoptosis of various cells, including endothelial and epithelial cells. Apoptotic turnover results in the release of intermediate cellular filaments such as cytokeratin-18 (CK-18). CK-18 is a major component of intermediate filaments and is widely expressed in epithelial and endothelial tissues. In apoptotic cells, CK-18 is phosphorylated and microfilaments are rapidly added. Different conditions of cellular stress increase the reorganization of cytoplasmic microfilaments while altering the polymerization of CK-18. In

Consequently, excision mediated by CK-18 caspases occurs during apoptosis and induces the formation of a specific neoepitope, recognized by the M30 antibody. The binding specificity of this antibody is

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restricts products of apoptotic degeneration of CK-18 (CK-18 cleaved by caspases [ccCK-18]). Interestingly, the presence of soluble ccCK-18 was associated with impaired lung function according to the COPD classification using the GOLD guidelines (fig. 6C).

5 The above data indicate an apoptosis dependent on pulmonary degeneration in serum samples. Excision of cytokeratin-18 and the release of ccCK-18 into the bloodstream occurs with disease progression and can be used to control patient morbidity.

Previous studies have demonstrated a predominance of the TH1 cytokine environment in patients with COPD. Since inflammation is always accompanied by the secretion of proteins known to have anti-inflammatory properties, such as IL-10, another protein that is known to be related to the innate immune system called interleukin-1 T1 receptor 4 was studied. This soluble molecule is considered to modulate the immune responses associated with TH1 / TH2. A significant increase in serum levels of the anti-inflammatory interleukin-1 receptor 4 was observed in patients with stages I and II COPD, but not in severe COPD. These

15 observations indicate that COPD shows an increase in innate immune activation markers and regulation similar to those of autoimmune diseases, sepsis and specific lung diseases such as fibrosis and asthma.

In recent work it has been shown that Toll-like receptors (TLRs) act as mammalian recognition pattern receptors that signal the presence of microbial components in cells of the innate immune system. At least 10 members of the mammalian TLR family have been published, each TLR showing a different specificity by molecular patterns of microbes. The binding of activation ligands, for example lipolysaccharide [LPS], to TLR induces the activation of NF-κB, which induces the production and release of proinflammatory cytokines. It is generally accepted that pre-exposure to LPS reduces sensitivity to a

25 second exposure to LPS, resulting in a decrease in the production of numerous cytokines in both rodents and humans. The molecular mechanisms of tolerance induced by TLR may involve regulation by decrease of the TLR4-MD2 complex that may lead to decreased TLR signaling. Additionally, it was demonstrated that the expression of IRAK-M and IRAK-1 as well as of the cytokine signaling suppressor (SOCS-1) is related to endotoxin tolerance.

Several studies assume an important role for the interleukin-1 receptor 4, a member of the Toll / IL-1 (TIR) receptor family, in the induction of endotoxin tolerance. ST2 is expressed in TH2 cells, mast cells and macrophages. It was shown that it is regulated by increase after stimulation with LPS and regulates cytokine expression in a model of acute lung injury. The secretion of the interleukin 1 receptor 4 in

35 The onset of COPD acts as a negative regulator of TLR4 and IL-1R signaling through MyD88 sequestration. In the clinical setting this may mean that the T1 / receptor 4 of the systemic interleukin-1 functions as a protective serum protein to prevent lung destruction due to an acquired and innate hyperactive immune system. It was important for this result the observation that T1 / receptor 4 of interleukin 1 is significantly correlated with serum levels of IL-10 in studies conducted in patient cohorts.

The results indicate that COPD patients show an increase in apoptotic turnover and the release of ccCK-18 and histone-DNA complexes into the systemic bloodstream. An increase in soluble T1 / receptor 4 of soluble interleukin-1 was also found in patients with stage I and II COPD compared to healthy smokers. Increased levels of receptor 4 of the anti-inflammatory interleukin-1 in all four

45 COPD stages may be a possible negative feedback loop to control the chronic inflammatory response. In addition, it was possible to demonstrate that soluble ICE correlates significantly with a history of smoking (pack-years) which indicates the triggering of inflammation due to harmful inhalant substances. The identified serum markers serve to identify patients at risk of development and progression of COPD.

EXAMPLE 2:

Material and procedures

55 patients

The set of patients used in this example is identical to the set of patients in example 1.

Thermal shock proteins 27, 60 and 70

The levels of HSP27, HSP60 and HSP70 were determined using enzyme-linked immunosorbent assay kits (ELISA) adapted for quantification of intracellular HSP (Douset IC, R&D Systems, USA). 96-well microtiter plates were coated overnight at 4 ° C with the capture antibody at a concentration of 1 µg / ml. After blocking the plates, serum samples and 65 standard proteins in different concentrations were added to the wells. After a washing step, biotin labeled antibody was added to each well and incubated for 1 hour. The plates were washed and streptavidin-HRP was added. The reaction of

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Color was achieved using tetramethylbenzidine (TMB; Sigma, USA) and stopped by the addition of an acid stop solution. The optical density was determined at 450 nm in an ELISA reader.

90 alpha heat shock protein

5 Serum levels of HSP90 alpha were measured with a commercially available ready-to-use ELISA kit (Stressgen, USA). Briefly, serum samples and standards were incubated in 96-well microtiter plates with anti-human HSP90 antibody. After a washing step, HRP-conjugated anti-HSP90 antibody was added and the plates were incubated for 24 hours. The plates were washed and TMB substrate was added. The color development was stopped by an acid stop solution and the optical density at 450 nm was determined. The amount of protein in each sample was calculated according to a standard curve of optical density values obtained from known levels of HSP90. The sensitivity of the ELISA was determined to be 50 pg / ml, the manufacturer established that the intra-assay variability was less than 10%.

15 Proteosome 20S

The microtiter plates were incubated overnight at 4 ° C with monoclonal antibody against the α6 subunit of the 20S proteasome (Biomol, USA). The plates were washed and blocked for 1 hour with 1% BSA in phosphate buffered saline. Serum samples and different concentrations of a standard protein (Biomol) were added, then the plates were sealed and incubated for 24 hours at 4 ° C. A rabbit polyclonal antibody against the α / β subunits of the 20S proteasome was added (Biomol) which served as a detection antibody and, after a washing step, the plates were incubated with a peroxidase-labeled rabbit anti-IgG donkey antibody (Jackson ImmunoResearch, United Kingdom) for another 2 hours. Tetramethylbenzidine was used as a color substrate. The reaction was stopped by adding 1 N sulfuric acid. The plates

25 were read at 450 nm using a Wallac Multilabel 1420 counter (PerkinElmer, USA).

Interleukin -6

Serum levels of IL-6 were determined by a commercial ELISA kit (BenderMedSystems, Austria). The tests were performed according to the manufacturing instructions. The plates were read at 450 nm in an ELISA reader, and the IL-6 content was calculated by comparing the optical density values of the samples with the optical density values of known IL-6 concentrations.

Statistical methods

35 The SPSS software (SPSS Inc., USA) was used to calculate all the results. A p value <0.05 was considered statistically significant. Peer comparisons were made between groups using the Mann-Whitney U test. Correlations were calculated using Spearman's correlation coefficient.

Results

HSP27

Serum HSP27 levels were 2042.57 pg / ml [1599.58-2485.57] (mean [95% confidence interval]) in

45 healthy controls, 2199.64 [1641.52-2757.75] in healthy smokers, 2862.62 [2280.49-3444.74] in COPD in stages GOLD I-II and 3717.58 [3079.35- 4355.81] in COPD in stages GOLD III-IV. Statistically significant differences were found between healthy controls and COPD in stages I-II (p = 0.025), healthy controls and COPD in stages III-IV (p <0.001), healthy smokers and COPD in stages III-IV ( p = 0.001) and COPD in stages I-II and COPD in stages III-IV (p <0.001) (fig. 7a).

HSP60

Serum HSP60 levels were 1836.69 pg / ml [153.30-3520.08] in healthy controls, 4378.40 [-3851.4812608.28] in healthy smokers, 3497.42 [-1561.38- 8556.23] in COPD in stages GOLD I-II and 531.81 [132.57

55 931.05] in COPD in stages GOLD III-IV. No statistically significant differences were found between the groups (Fig. 7b).

HSP70

Serum HSP60 levels were 140.50 pg / ml [67.97-213.04] in healthy controls, 108.50 [43.30-173.69] in healthy smokers, 454.29 [327.05- 581.52] in COPD in GOLD stages I-II and 437.92 [143.41-732.42] in COPD in stages GOLD III-IV. Statistically significant differences were found between healthy controls and COPD in stages I-II (p <0.001), healthy controls and COPD in stages III-IV (p = 0.009), healthy smokers and COPD in stages III-IV ( p <0.001) and healthy smokers and COPD in stages III-IV (p <0.001) (fig. 7c).

65

HSP90 alpha

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Serum HSP90 alpha levels were 13 133.78 pg / ml [9791.40-16 476.15] in healthy controls, 12 827.91 [10 838.21-14 817.62] in healthy smokers, 17 884 , 50 [13 307.14-22 461.85] in COPD in GOLD I-II stages and 17 273.02 [12 573.96-21 972.08] in COPD in GOLD III-IV stages. Statistically differences were found.

5 significant between healthy controls and COPD in stages I-II (p = 0.025) and healthy controls and COPD in stages III-IV (p = 0.049) (fig. 7d).

20S Proteosome

Serum levels of 20S proteasome were 194.78 ng / ml [164.94-224.62] in healthy controls, 188.25 [159,26217.25] in healthy smokers, 172.33 [133.78-210 , 88] in COPD in stages GOLD I-II and 187.50 [145,54-229,46] in COPD in stages GOLD III-IV. No statistically significant differences were found between the groups (Fig. 7e).

15 Interleukin -6

Serum levels of IL-6 were 5.18 pg / ml [0.17-10.19] in healthy controls, 1.65 [-0.11-3.42] in healthy smokers, 7.14 [1 , 19-13.09] in COPD in stages GOLD I-II and 2.99 [0.99-5.00] in COPD in stages GOLD III-IV. A statistically significant difference was found between healthy smokers and stage I-II COPD (p = 0.017).

Correlations and regression models

The correlations between HSP, FEV1% CV and IL6 are presented in Figures 8a to 8d. In regression models

25 unifactorial logistics including only healthy smokers and patients with COPD, HSP27 had an area under the curve (AUC) in the receiver operating characteristic curve (ROC) of 0.763 (0.624-0.902: 95% CI; p = 0.004) and HSP70 showed an AUC of 0.885 (0.786-0.983: 95% CI; p <0.001). None of the other variables showed significant results in the unifactorial logistic regression analysis (fig. 9).

Conclusion:

Patients with chronic obstructive pulmonary disease have progressive inflammation of the bronchial airways, small airways and pulmonary parenchyma. Pulmonary biopsies showed massive infiltration of peribronchial tissue with neutrophils, macrophages and lymphocytes as part of the immune system.

35 innate and acquired. Activation of these cells is considered to lead to remodeling of lung tissue. The slow progressive process of tissue remodeling is accompanied by a significant amount of cell destruction. In COPD, it is thought that both endogenous factors such as neutrophils and cytotoxic T cells, as well as exogenous stimuli such as tobacco smoke, contribute to tissue destruction. However, quitting smoking does not alter the insufficiency of the inflammatory response.

The induction of inflammatory signals and the increase in cell turnover result in the regulation by increased intracellular heat shock proteins and an increased release to the extracellular environment. In this example, a significant increase in HSP27 could be demonstrated in serum samples obtained from the peripheral blood flow of patients suffering from COPD compared to healthy smokers. HSP27 works as a repair mechanism that aims at the stability and correct post-translational folding of intracellular proteins as well as the prevention of apoptotic cell death. Elevated serum levels of HSP27 were reported in inflammatory disorders such as acute coronary syndrome and chronic transplant nephropathy. The expression of HSP27 is induced transiently in response to stress events. The termination of acute triggering results in regulation by immediate decrease of HSP27 concentrations to normal levels. Therefore, HSP27 is regulated by increment only when its cytoprotective properties are required. Interestingly, the results showed a continuous increase in serum concentrations of HSP27 with the intensity of the disease and a strong correlation with serum pro-inflammatory IL-6. This effect may be due to an increase in tissue devastation specifically in the late stages of COPD and the spread of inflammatory disease to other organic systems leading to

55 to a systemic spill of HSP27 into the vascular bed. HSP27 generally acts as an antiapoptotic mediator and can be considered an endogenous immunosuppressant that aims to control excessive inflammation in the clinical setting of COPD. Additionally, the serum content of HSP27 showed high sensitivity and specificity to determine the occurrence of COPD in a logistic regression model.

The role of extracellular HSP60 has not been well defined. The data provided in this document does not provide any support for HSP60 being a key element in the pathogenesis of COPD. Serum concentrations of HPS60 did not correlate with the levels of other HSPs.

Serum HSP70 levels were elevated in patients in early and late GOLD stages of COPD. Be

65 showed a four-fold increase in the GOLD I-II group compared to non-symptomatic smokers. HSP70 is an intracellular carbine molecule that is released into the extracellular space after cell death or by various

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secretion routes It has been reported that extracellular HSP70 activates innate and acquired immune system cells and stimulates cytokine production. An increase in soluble HSP70 concentrations was found in COPD samples. The maximum values in the COPD group in stages I-II indicate an enormous state of immune activation mainly in the initial stages of the disease. Additionally, HSP70 levels

5 correlated significantly with the levels of HSP27, HSP90 alpha and IL-16 and are therefore part of the systemic "danger signal" that accompanies immune activation in COPD. Additionally, the serum content of HSP27 showed high sensitivity and specificity to determine the occurrence of COPD in a logistic regression model.

10 Soluble alpha HSP90 was regulated by a significant increase in the peripheral bloodstream in groups with COPD compared to healthy non-smoking subjects. Elevated levels of HSP90 alpha have been previously described in aortocoronary bypass graft with pump and wound healing after hypoxia. HSP90 was also characterized as a central factor in the presentation of antigen to T lymphocytes through the molecules of the major class II histocompatibility complex (MHCII). As a summary of the data, it

15 concludes that high levels of extracellular HSP90 alpha in COPD are an essential stimulant of the acquired immune system, triggering a possible auto-reactive response to autoantigens. This function of HSP90 alpha can also change the immunogenicity of the associated antigen. Therefore, HSP90 alpha has immunomodulatory effects through cross-presentation of associated peptides in the context of the major histocompatibility complex molecules.

20 The extracellular serum content of the 20S proteasome did not decrease or increase statistically in the study cohorts investigated. The levels remained below 200 ng / ml in all groups and seemed to have secondary importance in the progression of COPD.

In summary, high serum concentrations of heat shock proteins 27, 70 and 90 alpha soluble immune modulators were shown in patients with COPD. This spillage in the vascular bed is at least partially caused by the continuous activation of the immune system in the deterioration of COPD through endogenous and exogenous trigger mechanisms. Additionally, HSP27 and HSP70 showed excellent statistical trends to serve as diagnostic markers for the detection of airway obstruction.

30 respiratory in initial stages of COPD.

Claims (10)

E11159730 09-29-2014 1. Procedure for the diagnosis of chronic obstructive pulmonary disease (COPD) in a human subject comprising the stages of: 5 -determine the amount of heat shock protein 27 (HSP27) in a sample of blood, serum or plasma from a human subject, -diagnosing COPD when the amount of HSP27 is increased compared to the amount of HSP27 in healthy human subjects. 2. Method according to claim 1, characterized in that the amount of interleukin-1 receptor 4, histone-associated DNA fragment, heat shock protein 70 (HSP70) and / or heat shock protein 90 alpha ( HSP90 alpha), in which COPD is diagnosed when the amount of receptor 4 of the Interleukin-1, histone-associated DNA fragment, HSP70 and / or HSP90 alpha is increased compared to the amount of interleukin-1 receptor 4, histone-associated DNA fragment, HSP70 and / or HSP90 alpha in human subjects healthy. 3. Method according to claim 1 or 2, characterized in that the amount of interleukin-1 receptor 4, histone-associated DNA fragment, thermal shock protein 27 (HSP27), thermal shock protein 70 (HSP70) and / or 90 alpha heat shock protein (HSP90 alpha) is determined by an immunoassay selected from the group consisting of enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and immunoblot. Method according to claim 2 or 3, characterized in that the amount of interleukin-1 receptor 4 in blood samples of a healthy human subject preferably ranges between 50 and 150 pg / ml, preferably between 60 and 140 pg / ml, more preferably between 70 and 130 pg / ml.

5.

Method according to claim 2 or 3, characterized in that the amount of histone-associated DNA fragment in blood samples of a healthy human subject is 20 to 50% less, preferably 25 to 40% less, than in blood samples of a human subject suffering from COPD.

6.

Method according to any one of claims 1 to 5, characterized in that the amount of protein

Heat shock 27 (HSP27) in blood samples of a healthy human subject ranges between 1500 and 2000 pg / ml, preferably between 1600 and 2400 pg / ml, more preferably between 1700 and 2300 pg / ml.

7.

Method according to claim 2 or 3, characterized in that the amount of heat shock protein 70 (HSP70), in blood samples of a healthy human subject ranges between 50 and 200 pg / ml, preferably between 70 and 190 pg / ml, more preferably between 80 and 180 pg / ml.

8.

Method according to claim 2 or 3, characterized in that the concentration of heat shock protein 90 alpha (HSP90 alpha), in a blood sample of a healthy human subject ranges between 10,000 and 15,000 pg / ml, preferably between 11,000 and 14,500 pg / ml, more preferably between 12,000 and 14,000 pg / ml.

Method according to any one of claims 1 to 8, characterized in that COPD is diagnosed when the amount of interleukin-1 receptor 4, DNA fragment associated with histones, HSP27, HSP70 and / or HSP90 alpha in the Sample is at least 10%, preferably at least 20%, increased compared to the amount of interleukin-1 receptor 4, DNA fragment associated with histones, HSP27, HSP70 and / or HSP90 alpha in healthy human subjects. Method according to any one of claims 1 to 8, characterized in that the risk of developing COPD is diagnosed when the concentration of receptor 4 of interleukin-1, histone-associated DNA fragment, HSP27, HSP70 and / or HSP90 alpha in the sample is at least 10%, preferably at least 20%, reduced compared to the concentration of receptor 4 of interleukin-1, DNA fragment associated with 55 histones, HSP27, HSP70 and / or HSP90 alpha in healthy human subjects. 11. Procedure to discriminate between COPD in stages I / II and COPD in stages III / IV in a human subject comprising the stages of: -determine the amount of term 27 shock protein (HSP27) in a sample of blood, serum or plasma from a human subject suffering from COPD, -diagnosing COPD in stages I / II when the amount of HSP27 is reduced compared to the amount of HPS27 determined in a sample of a human subject suffering from COPD in stages III / IV, or 65 -diagnosing COPD in stages III / IV when the amount of HSP27 is increased compared to the fifteen E11159730 09-29-2014 amount of HPS27 determined in a sample of a human subject suffering from COPD in stages I / II. 12. Method according to claim 11, characterized in that the amount of receiver 4 is also determined of interleukin-1, cytokeratin-18 cleaved by caspases (ccCK-18), histones and / or heat shock protein 5 70 (HSP70), in which COPD is diagnosed in stages I / II when the amount of receptor 4 of interleukin-1 or HSP70 is increased compared to the amount of interleukin-1 or HSP70 receptor 4 determined in a sample of a human subject suffering from stage III / IV COPD and / or when the amount of ccCK-18 or DNA fragment associated with histones is reduced compared to the amount of ccCK-18 or histone associated DNA fragment determined in a sample of a human subject suffering from stage III / IV COPD, 10 or in which stage III / IV COPD is diagnosed when the amount of interleukin-1 or HSP70 receptor 4 is reduced compared to the concentration of interleukin-1 or HSP70 receptor 4 determined in a sample of a human subject suffering from COPD in stages I / II and / or when the amount of ccCK-18 or histones is increased compared to the concentration of ccCK-18 or histone-associated DNA fragment determined in a sample of a human subject that you have COPD in stages I / II. 16